Oct 10, 2017 - mans affected with orofacial clefts. [22-â25], Down's ..... Lajeunie E, Le ... (1957)The ... 24. Human tooth-âsize asym-â metry in cleft lip with or without cleft palate. ..... Jung H,. Woo EJ, Von Cramon-â. Taubadel N. Amer J Phys.
Vol 5, No 1 (2017) ISSN 2167-‐8677 (online) DOI 10.5195/d3000.2017.78
Fluctuating dental asymmetry in rabbits with familial nonsyndromic coronal suture synostosis 1
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Mae A. Hyre , Seth M. Weinberg , Gregory M. Cooper , James Gilbert , Michael I. Siegel , Mark P. Mooney
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Oro-‐Maxillo-‐Facial Surgeon, Private Prac+ce, Charleston WV University of Pi.sburgh, School of Dental Medicine 3 University of Pi.sburgh, Department of Plas0c Surgery 2
Abstract Fluctua'ng dental asymmetry has been linked to condi'ons of unstable pre-‐ and peri-‐natal development. Familial, nonsyndromic craniosynostosis disrupts early craniofacial development through localized excessive calvarial ossifica-‐ !on leading to the premature fusion of the calvarial sutures. Such abnormal gene expression may also produce sys-‐ temic stress resul+ng in developmental instability, thereby affec+ng normal trait symmetry. The present study was designed to test this hypothesis by examining fluctua4ng dental asymmetry in an inbred strain of rabbits with familial, nonsyndromic coronal craniosynostosis. The mesiodistal (MD) and buccolingual (BL) dimensions of the right and le: maxillary first molars were measured in four groups of New Zealand white rabbits (N=176; n=40 with early-‐onset synostosis, n=65 with delayed-‐onset synostosis, n=46 in-‐colony, phenotypically normal rabbits, and n=25 wild-‐type normal controls). For each variable, raw signed asymmetry was calculated (le:-‐right) and tested for assump2ons of fluctua&ng asymmetry (i.e., normality and non-‐direc&onality). Any group that did not meet these assump.ons was excluded from further analysis. Using a standard size-‐adjusted, fluctua,ng asymmetry index, mean fluctua,ng asym-‐ metry was calculated and compared across groups with non-‐parametric sta+s+cs. For the MD dimension, no signifi-‐ cant (p > 0.05) group differences in mean fluctua8ng asymmetry were observed among groups. In contrast, rabbits with early-‐onset synostosis had significantly (p < 0.05) more fluctua;ng asymmetry in the BL dimension compared to wild-‐type controls. Results demonstrate increased fluctua1ng dental asymmetry in rabbits with nonsyndromic, early-‐ onset coronal suture synostosis and suggest that the molecular events producing suture synostosis locally may have also have systemic effects. Knowledge of these systemic interac/ons may contribute to a fuller understanding of the phenotypic spectrum observed in individuals with nonsyndromic craniosynostosis.
Cita%on: Hyre M, et al. (2017) Fluctua'ng dental asymmetry in rabbits with familial nonsyndromic coronal suture synostosis. Den$stry 3000. 1:a001 doi:10.5195/d3000.2017.78 Received: July 11, 2017 Accepted: August 18, 2017 Published: October 10, 2017 Copyright: ©2017 Hyre M, et al. This is an open access ar!cle licensed under a Crea!ve Com-‐ mons A"ribu"on Work 4.0 United States Li-‐ cense. Email: mpm4@pi&.edu
Introduction Nonsyndromic, simple cranio-‐ synostosis is a relatively rare condi-‐ tion (0.06 – 1.9/1000) that occurs when one or more of the calvarial sutures fuses prematurely during the period of active brain growth, result-‐ ing in aberrant patterns of growth in both the neurocranium and viscer-‐ ocranium [1]. In recent years, cranio-‐ facial biologists have begun to unrav-‐ el the biological pathways underlying abnormal suture morphogenesis. The emerging picture is one of complexi-‐ ty, involving multiple osteogenic growth factors and genes (FGFs, TGFβs, BMPs, MSXs, etc.) operating at various levels of regulation [2,3]. While the genetic mutations respon-‐ sible for syndromic cases of cranio-‐
synostosis have been known for some time [4,5], only recently have specific mutations in a number of genes (FGFR3 and MSX2 for e.g.) been iden-‐ tified in individuals affected with non-‐ syndromic coronal suture synostosis [5-‐8]. The number of familial cases, however, remains relatively small at around 14% for coronal cases and 6% for sagittal cases [1, 9,10], suggesting that sporadic mutations typically are involved. While significant progress has been made in elucidating the molecu-‐ lar causes of premature suture fusion, our understanding of the disease at the phenotypic level still remains in-‐ complete. In particular, it is unclear how alterations at the genotypic level are translated into the range of varia-‐
ble phenotypes that present under the heading nonsyndromic cranio-‐ synostosis. The presence of certain environmental exposures (e.g. ciga-‐ rette smoke), systemic factors (e.g. circulating levels of thyroxin or an-‐ drogens) and/or the additive effects of modifier genes may all act to mod-‐ ulate phenotypic severity [1]. There-‐ fore, given a genetic predisposition for nonsyndromic craniosynostosis, the specific phenotypic outcome may depend in part on the presence of additional deleterious factors, both intrinsic and extrinsic to the organ-‐ ism. The presence of such factors dur-‐ ing ontogeny may create a develop-‐ mental environment characterized by amplified stress, leading to both in-‐ creased synostotic severity and, pos-‐ sibly, other phenotypic manifesta-‐
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Fluctua'ng dental asymmetry in rabbits with familial nonsyndromic coronal suture synostosis
Vol 5, No 1 (2017) DOI 10.5195/d3000.2017.78
tions. Unfortunately, little is known about the phenotypic correlates of nonsyndromic craniosynostosis apart from the well-‐documented secondary growth effects on the skull. However, because many of the growth factors involved in the regulation of suture morphogenesis have expression pat-‐ terns in a variety of other tissues throughout the developing organism [1,2-‐5,11,12] it is unlikely that the extent of the disruption in nonsyn-‐ dromic craniosynostosis is limited entirely to the suture site. Increased stress during de-‐ velopment, either of genomic or envi-‐ ronmental origin, has been linked to specific phenotypic patterns in organ-‐ isms. One of the most commonly used phenotypic indicators of devel-‐ opmental stress is the degree of ran-‐ dom asymmetry present in normally symmetrical, paired traits, commonly referred to as fluctuating asymmetry [13]. Mather [14] reasoned that be-‐ cause bilateral characteristics are programmed by the same genes, ran-‐ dom deviations from symmetry will represent a phenotypic record of the
level of instability present during de-‐ velopment. Waddington [15,16] used the term canalization to refer to the degree to which genetically pro-‐ grammed developmental processes are able to resist perturbations during ontogeny. Developmental instability can be understood as the degree to which developmental processes are unable to buffer against such pertur-‐ bations, the consequences of which are manifested in the phenotype [17,18]. Interest in developmental in-‐ stability as a mechanism for elucidat-‐ ing the complex relationship between genotype and phenotype has in-‐ creased dramatically over the last decade [18-‐20]. Importantly, elevated levels of fluctuating asymmetry have been associated with a variety of stressors during development, includ-‐ ing a number of genetic diseases [21]. For example, there is evidence of ele-‐ vated fluctuating asymmetry in hu-‐ mans affected with orofacial clefts [22-‐25], Down’s syndrome [26,27], Fragile-‐X syndrome [28], premature birth [29,30] attention deficit-‐
Figure 1. Superior view of skulls from a 42 day wild-‐type normal control rabbits and rabbits with late-‐onset (postnatal) and early-‐onset (prenatal) coronal suture synostosis. Note the shorter, wider cranial vaults in the rabbits with coronal suture synostosis compared to the wild-‐type rabbit skull.
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hyperactivity disorder [31], general-‐ ized mental retardation [32], spinal malformation [33] and a wide range of psychiatric diseases including schizophrenia, major depression and bipolar disorder [21,34]. Because nonsyndromic craniosynostosis is a disease that disrupts craniofacial de-‐ velopment through abnormal calvari-‐ al bone homeostasis resulting in ex-‐ cessive ossification and premature suture fusion [2,3,35-‐37], affected organisms likely experience increased systemic stress during ontogeny. In principle, such stress should manifest itself in the phenotype as elevated levels of fluctuating asymmetry. However, De Leon and Richtsmieir [38] reported little fluctuating asym-‐ metry in the cranial vault bones of children with nonsyndromic sagittal suture synostosis and deserves fur-‐ ther study. Given the overlap in mo-‐ lecular expression between calvarial suture and dental morphogenesis [39], the association between dental abnormalities and syndromic forms of craniosynostosis [40], and the fact that both syndromic and nonsyn-‐ dromic cases may share a common genetic basis [5-‐8], it is reasonable to suspect that the teeth of nonsyn-‐ dromic craniosynostotic organisms develop within a stressed physiologi-‐ cal environment and may be a prime candidate for manifesting elevated levels of fluctuating asymmetry. In order to test the hypotheses that ge-‐ nomic stress is elevated in the pres-‐ ence of nonsyndromic craniosynosto-‐ sis, and that such stress increases with the severity of the cranio-‐ synostotic phenotype, we propose to investigate patterns of fluctuating dental asymmetry in a rabbit model with variably expressed, familial, non-‐ syndromic, coronal suture synostosis under controlled laboratory condi-‐ tions.
Fluctua'ng dental asymmetry in rabbits with familial nonsyndromic coronal suture synostosis
Vol 5, No 1 (2017) DOI 10.5195/d3000.2017.78
Materials and Methods Study sample Our study sample consisted of crani-‐ odental skeletal material from 176 New Zealand White rabbits (Oryctolagus cuniculus) affected with varying degrees of familial, nonsyn-‐ dromic, primary coronal suture synostosis. The rabbits were part of a unique breeding colony, established in 1993, at the University of Pitts-‐ burgh [41-‐46]. It has served as a use-‐ ful model for the study of familial, nonsyndromic craniosynostosis in human infants [35, 41-‐47]. All crania were obtained from rabbits enrolled in ongoing research protocols. All protocols were reviewed and ap-‐ proved by the University of Pitts-‐ burgh, Institutional Animal Care and Use Committee (IACUC). Rabbits from this colony ex-‐ hibit a wide range of phenotypes, in-‐ cluding: phenotypically normal carri-‐ ers; rabbits with delayed-‐onset synostosis (presenting at about 42 days post-‐natal), and; rabbits with early-‐onset complete unilateral or bilateral synostosis (presenting at about 21 days of gestation) [37, 41-‐ 45] (Figures 1 and 2). It has been shown recently that rabbits with ear-‐ ly-‐onset coronal suture synostosis (i.e., the most severe phenotype) were the most inbred and exhibited the highest coefficients of inbreeding [46]. For the present study, dentition from the skulls of 40 rabbits with ear-‐ ly-‐onset synostosis, 65 rabbits with delayed-‐onset synostosis, and 46 in-‐ colony, phenotypically normal rabbits were analyzed. Further, 25 wild-‐type rabbits of the same breed were in-‐ cluded as a separate control group. Table 1 shows basic descriptive statis-‐ tics of the sample by group. No dif-‐ ference in the distribution of males and females across groups was ob-‐ http://dentistry3000.pitt.edu
served. Thus, rabbits of both sexes were pooled during analysis. Further, due to sam-‐ ple size limitations, bilateral and unilateral affected animals were combined for all anal-‐ yses. All rabbits were housed and fed under similar labora-‐ tory conditions. Measurement procedure The mesiodistal (MD) and buccolingual (BL) dimen-‐ sions of the right and left first maxillary molars were meas-‐ ured with a set of Mitotoyo digital calipers (Japan) (Figure 3). The first maxillary molar was chosen because evidence suggests it is a very stable tooth developmentally [48]. This conservative approach Figure 2. Superior view of the coronal suture from a should bias our results against 42 day wild-‐type normal control rabbits and rabbits finding fluctuating asymmetry, with late-‐onset (postnatal) and early-‐onset (prena-‐ thus minimizing the chances of tal) coronal suture synostosis. Note the obliterated reporting a false-‐positive. For the coronal suture in the early-‐onset synostosis rabbit skull (bottom picture), the dysmorphic and incom-‐ MD measurement, the arms of plete coronal suture in the late-‐onset synostosis the calipers were positioned at rabbit skull (middle picture), compared to a normal the widest portion of the tooth coronal suture in the wild-‐type rabbit skull (top at the occlusal surface (Figure 3). picture). For the BL measurement, one arm of the calipers was positioned Following standardized flush against the buccal edge of the methodological guidelines [49,50], tooth. With the one arm in place, the the statistical nature of the asym-‐ measurement was then taken across metry was explored prior to further the tooth at the level just below the analysis. For fluctuating asymmetry, occlusal plane on the lingual edge two conditions must be met. The raw, (Figure 3). The BL measurement was signed left-‐right difference scores not taken directly at the occlusal sur-‐ must have both: 1) a normal distribu-‐ face in order to avoid measuring the tion and 2) a mean equal to zero. The effects of functional wear. All meas-‐ first assumption was tested with the urements were taken by one individ-‐ Kolmogorov-‐Smirnov test for normali-‐ ual (MAH). ty, while a one sample t-‐test was per-‐ formed to determine whether the Statistical analysis mean of the signed asymmetry scores Testing assumptions of fluc-‐ is significantly different from zero. A tuating asymmetry: mean significantly different from zero indicates directional asymmetry, which may have different biological
Fluctua'ng dental asymmetry in rabbits with familial nonsyndromic coronal suture synostosis
Vol 5, No 1 (2017) DOI 10.5195/d3000.2017.78
implications than fluctuating asym-‐ metry. Tests of fluctuating asymmetry assumptions were carried out for each variable within each group sepa-‐ rately. For both MD and BL varia-‐ bles, the phenotypically normal rab-‐ bits were the only group that failed the test of normality (p < 0.05). The phenotypically normal (in-‐colony) control group was therefore dropped from subsequent analyses. For the BL variable, mean raw asymmetry in the delayed-‐onset group was significantly different from zero (t = 3.869; p < 0.001), indicating the presence of di-‐ rectional asymmetry. As a result, the delayed-‐onset group was dropped from further analysis of the BL varia-‐ ble. Because random measure-‐ ment error is statistically identical to fluctuating asymmetry, the degree of error present in the data must also be assessed. Repeat measurements were obtained on all 176 animals by the same observer (MAH). For each variable, measurement error was quantified by compu-‐ ting the mean absolute differ-‐ ence (MAD) from the replicate measures [51]: Mean Absolute Difference = ∑ |Time 1 – Time 2| / N This value was then compared directly against the standard unsigned mean fluc-‐ tuating asymmetry index: Fluctuating asymmetry = ∑ |Left – Right| / N If the magnitude of the mean absolute difference is determined to be less than that of mean fluctuating asymmetry for a given variable, this indi-‐ cates that residual fluctuating http://dentistry3000.pitt.edu
asymmetry exists even after meas-‐ urement error is taken into account. Conversely, if the mean absolute dif-‐ ference is greater than mean fluctuat-‐ ing asymmetry, then we cannot be certain that any measurable fluctuat-‐ ing asymmetry exists. For both varia-‐ bles in the present study, mean abso-‐ lute difference was less than mean fluctuating asymmetry (MD: 0.05 vs. 0.07; BL: 0.04 vs. 0.09). This pattern held true both overall and within each group separately, indicating that fluctuating asymmetry was not swamped by random measurement error. For some biological traits, in-‐ creased trait size is related to elevat-‐ ed levels of fluctuating asymmetry. Thus, the effects of size on fluctuating asymmetry magnitude must be con-‐ sidered. For each variable, Pearson correlation coefficients were calcu-‐ lated between original trait values and unsigned FA values (|Left – Right|). Because a weak but signifi-‐
cant correlation (r = 0.20; p < 0.01) was observed for the MD variable, an alternative size-‐adjusted FA index was used for subsequent group compari-‐ sons: Fluctuating asymmetry = (|Left – Right| / 0.5 Left + Right) Group fluctuating asymmetry comparisons: Using the standard size-‐ adjusted index, mean fluctuating asymmetry across groups was com-‐ pared using nonparametric statistics, followed by post-‐hoc comparisons where necessary. The nonparametric approach was employed because the fluctuating asymmetry index chosen for group comparisons converts left-‐ right differences into absolute values, which by nature are not distributed normally. Because certain groups failed to meet the assumptions of fluctuating asymmetry, certain group comparisons could not be carried out. For the MD variable, only three
Fluctua'ng dental asymmetry in rabbits with familial nonsyndromic coronal suture synostosis
Vol 5, No 1 (2017) DOI 10.5195/d3000.2017.78
groups (early-‐onset, delayed-‐onset, and wild-‐type controls) were com-‐ pared with a Kruskal-‐Wallis ANOVA. For the BL variable, only two groups (early-‐onset and wild-‐type controls) were compared, thus a Mann-‐ Whitney U t-‐test was used. Statistical analyses were performed with the program SPSS 11.5. All differences were considered significant if p < 0.05.
Results For the MD dimension, rab-‐ bits with early-‐onset synostosis had more fluctuating asymmetry than rabbits with delayed-‐onset synostosis while wild-‐type control rabbits had a greater magnitude of fluctuating asymmetry than either affected group. However, no significant (p > 0.05) group differences in mean fluc-‐ tuating asymmetry were observed for the MD dimension (Table 2). In contrast for the BL dimension, rabbits with complete early-‐onset synostosis had a significantly (p < 0.05) greater magnitude of fluctuating asymmetry than wild-‐type control rabbits (Table 2). Although mean age differed con-‐
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siderably across groups, no significant correlation (p> 0.05) between FA and age was observed for either tooth dimension.
Discussion Familial, nonsyndromic crani-‐ osynostosis is a disease characterized by abnormal calvarial bone regulation early in prenatal life [2,3,35-‐37]. It was hypothesized that this disruption in homeostasis would lead to in-‐ creased systemic physiological stress during the critical period of den-‐ togenesis, resulting in increased fluc-‐ tuating dental asymmetry. Results of the present study confirm this hy-‐ pothesis; rabbits with early-‐onset synostosis had a significantly greater magnitude of fluctuating dental asymmetry for the BL dimension of the first maxillary molars compared to unaffected controls. This pattern was not observed, however, for the MD dimension. These results suggest that the teeth of rabbits with congen-‐ ital nonsyndromic craniosynostosis develop within a stressed physiologi-‐
cal environment. Unfortunately, be-‐ cause certain groups did not satisfy the statistical requirements of fluctu-‐ ating asymmetry, all group compari-‐ sons were not possible. It could not be determined, therefore, whether fluctuating asymmetry was related to craniosynostosis in a severity-‐ dependent manner. These results, although limited in scope, suggest that a broader array of primary de-‐ fects may be present in individuals with nonsyndromic craniosynostosis, and that these may not be limited entirely to the suture site. In princi-‐ ple, a fuller understanding of the phenotypic correlates present in non-‐ syndromic craniosynostosis may shed light on disease etiology. For in-‐ stance, predictions can be generated about patterns of phenotypic varia-‐ tion from the known expression pat-‐ terns of specific candidate genes. Prior to the present study, dental fluctuating asymmetry had not been examined in individuals affected with craniosynostosis. Previous re-‐ search into dental development with-‐ in the craniosynostoses has been fo-‐ cused almost entirely on syndromic forms of the disease. In Apert syn-‐ drome, for instance, a wide variety of dental anomalies have been report-‐ ed; these include delayed develop-‐ ment, ectopic teeth, hypodontia and supernumerary teeth [52,53]. Similar-‐ ly, in cases of Crouzon syndrome, ec-‐ topic eruption, delayed development and hypodontia have all been ob-‐ served [54,55,12]. Pfeiffer syndrome may be characterized by natal teeth [56], suggesting precocious dental development. This is further support-‐ ed by reports of supernumerary and ectopic teeth [57]. Given the fact that FGFs play a role in both dental devel-‐ opment and suture morphogenesis, it is perhaps not surprising that the teeth are affected in cases of syn-‐
Fluctua'ng dental asymmetry in rabbits with familial nonsyndromic coronal suture synostosis
Vol 5, No 1 (2017) DOI 10.5195/d3000.2017.78
dromic craniosynostosis [39,11,58]. In light of this overlap in molecular ex-‐ pression, these conspicuous dental defects are most likely the result of aberrant FGF signaling within the de-‐ veloping dentition and its surround-‐ ing tissues [40].
in a number of naturally and experi-‐ mentally stressed human and non-‐ human populations, although the source of the stress is sometimes not well defined [24, 27, 28, 64-‐70].
A number of possible expla-‐ nations for the present findings may It remains unclear, however, be offered. One possibility is that the whether the observed pattern of den-‐ same genetic mutation that causes tal fluctuating asymmetry results premature suture fusion also results from such pleiotropic effects. Be-‐ in increased dental fluctuating asym-‐ cause fluctuating asymmetry is a ra-‐ metry. The genetic mutation in these ther generic response to stress during rabbits has been identified recently as development, we can only speculate belonging to the fgfr family of genes as to whether the underlying cause of working in tandem with a modifying gene (manuscript in preparation) so this may be a possibility; however, no obvious dental anomalies have been observed in this model, sug-‐ gesting that overall dentogenesis is probably not affect-‐ ed in these rabbits. Accumulating evi-‐ dence also suggests that altered calvarial bone regulation, Figure 3. Inferior view of the rabbit skull showing the post-‐ resulting in hyperos-‐ incisor maxillary dentition (P1-‐M3) (first three premolars and tosis and subsequent molars). The arrows on M1 (first molar) represent the mesi-‐ premature suture odistal and buccolingual dimensions that were measured in the fusion in cases of present study. nonsyndromic crani-‐ the fluctuating asymmetry is the osynostosis, stems from localized, same as that of the craniosynostosis. abnormal expression of various bone Dental dimensions have been shown regulating growth factors (e.g., BMPs, to be highly heritable and are thought FGFs, and/or TGFβs) [1-‐3, 36]. Such to be under relatively tight genetic abnormal growth factor expression control [59-‐61]. That being said, it is may also primarily affect other devel-‐ also clear that environmental influ-‐ oping antimeric osseous structures ences such as local and systemic resulting in fluctuating asymmetry. physiological factors, teratogens, and However, DeLeon and Richtsmieir extra-‐organismic variables play a crit-‐ [38] reported no significant fluctuat-‐ ical role in determining the final form ing asymmetry in the crania of 22 and size of teeth, especially during children with nonsyndromic sagittal the early stages of dental develop-‐ synostosis, although the effects could ment [62,63]. Increased fluctuating have been ameliorated with age and dental asymmetry has been reported http://dentistry3000.pitt.edu
remodeling. In addition, no significant changes in postcranial skeletal (e.g., first metacarpal) growth have been observed in synostotic rabbits [41-‐ 45], although fluctuating skeletal asymmetry has not been examined yet and this mechanism remains a possibility. Another possibility is that in-‐ creased intracranial pressure result-‐ ing from premature suture fusion could invoke a generalized stress re-‐ sponse by activating the hypothalam-‐ ic-‐pituitary-‐adrenal axis, which may increase levels of fluctuating calcium during dentogenesis [65,71], and sec-‐ ondarily result in increased dental fluctuating asymmetry in rabbits with early-‐onset synostosis. Increased fluc-‐ tuating asymmetry has also been linked to external environmental stressors such as heat, cold and noise [64,65,67]. However, these factors are unlikely to play a major role in this study because all the rabbits in the present sample were raised under similar controlled, environmental conditions. It is also unlikely that functional wear effects account for these results since such effects are typically associated with directional asymmetries and should increase with age (no association between age and fluctuating asymmetry was ob-‐ served). In addition, rabbit teeth are continually erupting and the lack of age related differences in dental fluc-‐ tuating asymmetry suggest that the stress associated with craniosynosto-‐ sis may be chronic in rabbits with ear-‐ ly-‐onset synostosis. Finally, canaliza-‐ tion of dental development may be under separate genetic control. Un-‐ der this premise, less canalized organ-‐ isms would be predisposed to mani-‐ festing elevated dental fluctuating asymmetry in the presence of delete-‐ rious factors, such as aberrant growth factor signaling. We recently reported
Fluctua'ng dental asymmetry in rabbits with familial nonsyndromic coronal suture synostosis
Vol 5, No 1 (2017) DOI 10.5195/d3000.2017.78
a significant, negative relationship between coronal suture growth and the coefficient of inbreeding [46] in rabbits from this colony. Rabbits with early onset synostosis showed the least growth and greatest amount of inbreeding. It is possible that addi-‐ tional modifier genes accumulated from inbreeding were responsible for increased systemic stress and the in-‐ creased levels of fluctuating dental asymmetry in these rabbits. Hueze et al. [72] also showed that human cra-‐ nia exhibiting a phenotypic continu-‐ um with skulls with bicoronal sutures synostosis displaying more intense asymmetry that skulls with unicoro-‐ nal shape which supports this hy-‐ pothesis. Unfortunately, the present study was not designed to test these competing hypotheses. Fluctuating asymmetry is a subtle marker of de-‐ velopmental disturbance and its per-‐ vasiveness both in the craniodental complex and other anatomical re-‐ gions of craniosynostotic organisms will need to be explored further in order to begin to unravel its biological basis. Given the numerous reports of various dental defects associated with syndromic craniosynostosis, it may be worthwhile to investigate other aspects of the dentition in non-‐ syndromic synostotic populations as well. Acknowledgements The authors would like to thank Kate Quinlin for her assistance in the lab. This work was supported in part by a grant from NIH/NIDCR (P60 DE13078).
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